A key challenge in developmental biology is to reconstruct critical developmental pathways in vitro, which not only provides novel experimental opportunities but also serves as a basis for medical applications. In multicellular organisms, the germ cell lineage is endowed with the critical function of ensuring the creation of new organisms, thereby perpetuating the genetic and epigenetic information across the generations. The reconstruction of the development of the germ cell lineage in vitro is therefore of fundamental significance in the life sciences in general. There have been several attempts to generate gametes or their progenitors (primordial germ cells: PGCs) in vitro from embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of blastocysts in mice1,2,3,4,5 and humans 6,7,8,9,10,11 (see also the reviews in12,13,14). However, all these attempts involved random differentiation of ESCs as embryoid bodies under undefined conditions and relied on spontaneous expression of a marker gene(s). Consequently, these efforts were inefficient at obtaining the cells of interest. Moreover, the cells created have never been demonstrated to contribute to the generation of healthy offspring.
We have identified key transcriptional regulators, Blimp1 (also known as Prdm1) and Prdm14, for the specification of the germ cell lineage from the epiblast15,16, and uncovered a genome-wide transcriptional dynamics associated with PGC specification17. Recently, we defined a signalling principle for PGC specification from the epiblast and showed that essentially all the epiblast cells in mice from embryonic day (E) 5.5 to E6.0 but not those later than E6.25 were efficiently and reproducibly induced into Blimp1-, Prdm14-, stella-, and alkaline phosphatase (AP)-positive PGC-like cells by cytokines including BMP4 under serum- and feeder-free, defined conditions (serum-free medium: SFM) in a floating culture18. When transplanted into the testes of neonatal W/Wv mice lacking germ cells, the induced PGC-like cells underwent proper spermatogenesis and contributed successfully to the generation of the healthy offspring18. These findings demonstrate that an efficient and reproducible induction of PGCs with proper function from pluripotent stem cells such as ESCs or induced pluripotent stem cells (iPSCs) in culture, the essential first step of the reconstruction of germ cell development in vitro, may be achieved through initial differentiation of pluripotent stem cells into epiblast-like cells bearing properties of the pre-gastrulating epiblast cells at E5.5-E6.0. However, culture conditions capable of efficiently and reproducibly inducing the pre-gastrulating epiblast-like cells (EpiLCs) from pluripotent stem cells have not been found.